1. Field of the Invention
This invention relates to the two fields of genetic engineering and drug design and is more particularly related to the identification and preparation of drugs effective in inhibiting a particular purified enzyme, the enzyme being available as a result of genetic engineering of the gene that encodes the peptide.
2. Description of the Background
Pneumocystis carinii pneumonia is a leading cause of morbidity and mortality in acquired immunodeficiency syndrome (AIDS). Since the onset of the AIDS epidemic, the incidence of P. carinii pneumonia has risen from approximately 200 cases per year to much greater than 25,000 cases per year in the U.S. Due to the lack of a continuous in vitro culture system and the cumbersome nature of the rat model of P. carinii pneumonia, anti-P. carinii therapy has been developed largely on the assumption that anti-protozoan agents were likely to be effective. In fact, P. carinii has recently been shown to be a member of the Fungi.
The two principal therapeutic modalities, trimethoprim/ sulfamethoxazole and pentamidine, were developed using the anti-protozoan theory. Prior to the AIDS epidemic, these agents were sufficient for treatment of the rare cases of P. carinii pneumonia. However, in the HIV-positive patient, therapy and prophylaxis with the standard anti-P. carinii agents are complicated by frequent toxic and allergic side effects. New compounds active against P. carinii are clearly needed.
The inability to propagate P. carinii reliably in vitro and the limited quantities of P. carinii enzymes that can be purified from infected rat lungs have hindered the search for anti-P. carinii agents. Purification and characterization of the intracellular targets for such agents would enable the development of new therapies of P. carinii pneumonia.
Of the known anti-P. carinii agents, the dihydrofolate reductase (DHFR) inhibitors are the most thoroughly characterized. DHFR plays a central role in the de novo synthesis of nucleic acid precursors. DHFR inhibitors (e.g., methotrexate, trimethoprim, and pyrimethamine) are effective anti-neoplastic, anti-bacterial, and anti-protozoal agents. P. carinii pneumonia clearly responds to the combination of a DHFR inhibitor (trimethoprim or pyrimethamine) and a sulfonamide. However, despite their obvious efficacy when used in conjunction with a sulfonamide, trimethoprim and pyrimethamine are in themselves poor inhibitors of P. carinii DHFR [50% inhibitory concentration values (IC.sub.50) of 39,600 and 2,400 nM respectively compared to 8 and 2,500 nM for E. coli DHFR at similar substrate concentrations]. Other antifolates have been shown to be more effective inhibitors of P. carinii DHFR, but require concomitant administration of leucovorin to prevent host toxicity. If pure P. carinii DHFR were available for study, then inhibitors that surpass the efficacy of the known antifolates could be found by comparing binding affinities of the inhibitor to P. carinii DHFR versus mammalian DHFR.